Jul 14, 2008

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Bt maize and soil

Hot on the trail of transgenic molecules

Genetically modified Bt maize produces an insecticide called Bt toxin. Small quantities of this Bt toxin are released into the soil by the plants, especially from plant remains left on the field after harvesting. The effect of Bt toxin on the soil has been studied for several years in the laboratory of the Johann Heinrich von Thünen-Institut, Federal Research Institute for Rural Areas, Forestry and Fisheries (vTI). Christoph Tebbe and his team have adapted and refined a standard procedure which enables them to detect even tiny traces of Bt toxin in environmental samples. The results so far indicate that Bt concentrations in the soil are so miniscule that no negative effects on soil-dwelling organisms are to be expected.

ELISA (enzyme-linked immunosorbent assay) is a highly sensitive method of detecting Bt toxins. This technique uses antibodies which bind specifically to the Bt toxin of interest. The bonding triggers an enzymatic reaction, which in turn produces a colour reaction. This can be measured automatically and used to calculate the Bt toxin concentration. Several samples in varying dilutions (up to a maximum of 96 samples) can be measured simultaneously on the ELISA plates. From soil sample to protein solution

The soil samples and plant remains from the field have to go through a number of processing steps before the clear fluid which is examined in the ELISA test is obtained.

The leaf and root remains are removed from the soil samples, the soil is sieved and then mixed with a saline extraction buffer whilst being vigorously shaken, to release the proteins. To optimise protein extraction, the mixture is set aside for an hour at room temperature before being centrifuged. Because of the very low concentrations, the researchers have added an extra ultra-filtration stage, which results in a seventeen-fold concentration of the solution. The solution obtained is clear and can be used directly in the ELISA assay.

The plant material being investigated is washed first to remove the soil, then frozen in liquid nitrogen and ground in a mortar, before being mixed with the extraction buffer. Since the plant remains are expected to contain higher concentrations of Bt toxin than the soil samples, the additional filtration stage is omitted.

The refined ELISA test enables Christoph Tebbe and his team to detect Bt toxin to a limit of 0.01 nanograms per gram of soil. This can be likened to a single sugar lump in a cube measuring 33 metres cubed.

Bt toxin in the soil: Minute concentrations

The gene inserted in Bt maize which causes the plant to produce Bt toxin is derived from a bacterium called Bacillus thuringiensis. Although this bacterium is commonly found in soil, up until now it has not been possible to detect any Bt toxin in natural soils. This is because these bacteria are present mainly in the form of resting spores which contain a non-toxic, crystalline pre-stage of the Bt protein, rather than the free Bt toxin. The spores regularly germinate, and also multiply, in the gut of insects. However, they are only pathogenic for a few specific insect species (hosts).

Bt toxin acts very specifically to control certain chewing pests. Christoph Tebbe and his team have studied two different Bt toxins: Cry1Ab, which is effective against the European corn borer, and Cry3Bb1, which is toxic to the Western corn rootworm.

The highest Cry1Ab concentration recorded in rhizosphere soil was 1.9 nanograms per gram of soil based on dry weight. Bt maize had been grown on the soil under investigation for between one and three years. European corn borer larvae were found to contain more than 2 micrograms of Bt toxin. The LD50 (the lethal dose that kills 50 percent of the animals tested) for European corn borer larvae is between 1.5 and 2.4 micrograms per gram. In other words, the concentration required to affect the pest is around 1000 times higher than the highest amount recorded in the rhizosphere soil samples taken from the field.

Christoph Tebbe’s team detected Cry3Bb1 at a maximum concentration of 1.65 nanograms per gram of rhizosphere soil. Here too, the LD50 of around 1 microgram per gram for the Western corn rootworm, the target organism for the Cry3Bb1 Bt toxin, is far higher.

In view of the fact that these two maize pests are the most sensitive organisms to Cry1Ab and Cry3Bb1 respectively, it can be assumed that a soil concentration one thousand times smaller has no harmful effects on other organisms living in and on the soil. Another team found 0.2 micrograms of toxin in ground beetle larvae, which had no harmful effect.

Bt toxin is broken down

Christoph Tebbe is working on the assumption that the available detection sensitivity is sufficient as far as direct toxicity is concerned. But he points out we should not overlook the fact that sublethal effects can occur in non-target organisms, affecting reproduction rates, for example. These could potentially arise even at low concentrations, if the organisms are exposed to the toxin for extended periods of time. Bt toxins can break down relatively quickly, depending on the nature of the soil and climatic conditions. A fraction may persist for longer in the soil, but the toxin does not accumulate over the years, as is often feared.

Christoph Tebbe concludes from his research and from the findings of other safety research projects that, at the very least, the thoroughly investigated Mon810 Bt maize with the Cry1Ab Bt toxin can be classified as safe. He maintains that Bt maize is ecologically harmless, especially when compared with conventional insecticide treatments. But since the different Bt toxins have very different host spectra and furthermore are produced in varying concentrations by the transgenic plants in question, the scientist emphasises the need to examine and evaluate each toxin on a case-by-case basis. For example, the concentration of Cry3Bb1 in the plant cells of the maize variety under investigation is many times higher than that of Cry1Ab in MON810, and yet Cry3Bb1 breaks down much more quickly in the soil.

Cry 3Bb1
	Root	Leaf	Root after harvesting	Leaves after harvesting		Root soil	Bulk soil
Scale	100µg/g (10-4)	10 µg/g (10-5)	1 µg/g (10-6)	0,1 µg/g (10-7)	10 ng/g (10-8)	1 ng/g (10-9)	0,1 ng/g (10-10)
			LD 50 for the Western corn rootworm	no effect in ground beetles	no effect in sciarid fly larvae

Cry3Bb1 concentrations in different plant parts and soils

		Cry1AB (MON810)	Cry3Bb1 (MON88017)
ELISA detection limit		0,01 ng/g dry weight
	Root	10-20 µg/g (when the maize is in flower)	Approx. 100-370 µg/g
	Root after harvesting	3-8 µg/g (4 to 6 weeks after harvesting)(not differentiated by leaf/root)	On average 2 µg/gsix months after harvesting: 0.3 to 14 ng/g
	Leaves after harvesting		162 ng/g
	Root soil	Max. 1.9 ng/g average 0.4 ng/g (when the maize is in flower)	Max. 1.65 ng/g average below 0.05 ng/g0.06 ng/g (4 to 6 weeks after harvesting)
	Bulk soil	0.01 – 0.08 ng/g (4 to 6 weeks after harvesting)no longer detected 15 months after harvesting	Max. 0,05 ng/g
	LD 50 European corn borer	1,55 – 2,42 µg/g 4-100 ng/cm²
	LD 50 Western corn rootworm		0,3-1 µg/ml diet 10,4 µg/cm²

Comparison of Cry1Ab and Cry3Bb1 concentrations in different plant parts and soils, and LD50 values for the respective target organisms